Ebselen (Ebs) is considered as a glutathione peroxidase (GPx) mimetic and primarily thought to function by scavenging intracellular reactive oxygen species (ROS). Previous to our work, Deng et al. (2010a) demonstrated complete block of ICl,swell with 15 microM Ebs following endothelin-1 (ET-1) induced activation of the current in cardiomyocytes. This block was presumed to take effect mainly via the quenching of ROS. Nonetheless, our work with DI TNC1 astrocytes strongly emphasizes that Ebs might function by an alternative mechanism based on its kinetic profile in blocking ICl,swell. Our experiments showed that 45 nM Ebs can fully block ICl,swell thus suggesting an apparent IC50 result, we predicted Ebs to possess a high kon with a low koff close to zero. As predicted, Ebs failed to washout in the timescale covered by our patch-clamp experiments. The block was also distal to H2O2, previously considered as the most proximate regulator of ICl,swell. And based on further evidence demonstrating irreversible block of ICl,swell distal to H2O2 with Ebs congeners, complete suppression of native ICl,swell with MTS reagents, and failure of Ebs to block ICl,swell from the cytosol, we concluded that Ebs and its congeners can covalently modify important –SH groups required for current activation while functioning as sulfhydryl reagents. Complete irreversible block of ICl,swell with 110 mM cell impermeant MTSES in native DI TNC1 astrocytes contrasts sharply to SWELL1 (Qiu et al., 2014) or LRRC8A (Voss et al., 2014), the latest molecular entity presumably responsible for ICl,swell, where 3.33 mM MTSES failed to demonstrate block of ICl,swell in the wild-type stably expressing SWELL1 (Qiu et al., 2014). Our data with Ebs, its congeners, and MTS reagents indicate the existence of a common extracellular binding site which involves a selenenylsulfide (Se-S) bond that critically modulates ICl,swell. We, therefore, synthesized a derivative of Ebs called ebselen-para-yne (Ebs-p-yne), which provided an even higher affinity for blocking ICl,swell with a presumed IC50 ~picomolar range. Ebs-p-yne is a promising novel molecule that may serve as a tag in identifying the molecular fingerprint ultimately responsible for ICl,swell. Furthermore, we can take advantage of click chemistry to ultimately pull out the channel or channel component which has remained elusive for greater than two decades.

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ABSTRACT This thesis describes an investigation into the role of acetylenic and allenic precursors in the formation of the important aroma compound β-damascenone (1). Chapter 1 provides an introduction to the subject, beginning with a brief history of the Australian wine industry which began with the first fleet’s arrival in 1788. Many of the various volatile compounds found in wine are then discussed, with particular emphasis on β-damascenone (1). Some previous syntheses of 1 are summarised, as well as the in vivo generation of this compound, and also the role of glycoconjugation in nature. The chapter concludes with the aims of the present work. Chapter 2 covers the synthesis of the suspected acetylenic precursor 9-hydroxymegastigma-3,5-dien-7-yne (36), which was prepared by the addition of 3-butyn-2-ol to 2,6,6-trimethylcyclohex-2-en-1-one, followed by a conjugate dehydration reaction. The synthetic sample of 36 was shown to be identical to a compound previously observed in the hydrolysate of 3,5,9-trihydroxymegastigma-6,7-diene (31). Upon acid hydrolysis, 36 produced > 90% β-damascenone (1). Chapter 3 outlines the synthesis and hydrolysis of the C9 glycoside 43, which was prepared by a modified Koenigs-Knorr procedure on aglycone 36. Diastereomerically pure samples of each of the two possible glycosides were synthesised from corresponding enantiomerically pure samples of 36, which in turn were prepared by the use of either (R) or (S) 3-butyn-2-ol. Detailed hydrolytic studies (at 25 ºC) were conducted on both the aglycone and the two glycosides: the half lives of conversion of 36 into 1 were 40 hours and 65 hours at pH 3.0 and pH 3.2 respectively; the (9R) diastereomer of 43 had half-lives of 3 days and 6 days, respectively at the same pH values, whereas the (9S) diastereomer had half lives of 3.5 days and 6.5 days, respectively at the same pH values. The synthesis of the other suspected precursor, megastigma-4,6,7-triene-3,9-diol (35) is covered in Chapter 4. This allene was prepared by addition of 3-butyn-2-ol to phorenol, with the allene function generated by reaction with lithium aluminium hydride. By using (3S)-phorenol and both (R) and (S) 3-butyn-2-ol, four different diastereomers of 35 were prepared and characterised. The (3S, 6R, 9S)-isomer of 35 was also found to be identical to a compound previously observed in the hydrolysate of (31). A detailed hydrolytic study of the four synthetic isomers of 35 is contained within Chapter 5. This study revealed that each of the four isomers underwent rapid epimerisation at 25 ºC and pH 3.0. Careful analysis of the four product mixtures by chiral GC-MS revealed that this epimerisation was occurring exclusively at C3. The complete absence of 3-hydroxydamascone (2) from any of the hydrolysates required a re-appraisal of the mechanism of in vivo formation of β-damascenone (1), which forms the focus of the second half of this chapter. The experimental procedures (materials and methods) for all work covered in chapters 2-5 are located in Chapter 6.